4a). was downregulated particularly in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic manifestation AZD8055 of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in individuals with hepatocellular carcinoma, low manifestation of LHPP correlated with increased tumour AZD8055 severity and reduced overall survival. Thus, LHPP is definitely a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is definitely oncogenic. Liver malignancy is the second leading cause of cancer-related deaths globally. Hepatocellular carcinoma (HCC) represents approximately 90% of main liver cancer instances3. Recent studies indicate that almost 50% of HCC instances display aberrant PI3KCAKTCmTOR signalling4, including loss of the tumour suppressors PTEN, TSC1, and TSC23 (Fig. 1a). We generated an HCC mouse model by liver-specific deletion of PTEN and TSC1, using the neonatally indicated albumin promoter like a driver (see Methods). We hereafter refer to this model as liver-specific double-knockout (L-dKO) mice. L-dKO mice invariably exhibited hepatomegaly and advanced liver tumours at 6 and 20 weeks of age, respectively (Fig. 1b and Extended Data Fig. 1a). Histopathological analysis (Fig. 1b) and molecular classification based on mRNA manifestation of the liver malignancy markers (also known as and (= 12). 3,147 proteins were quantified in a minimum of 10 tumours, of which 433 proteins were upregulated (reddish), 262 were downregulated (blue), and 2,452 were unchanged (yellow). ANOVA-based two-sample = 12 for NME1, NME2, LHPP and PHPT1; = 9 for PGAM5) compared to control livers (= 6); data are mean s.d. PHPT1 and PGAM5 are two defined mammalian histidine phosphatases. We performed quantitative proteomic analysis on three tumours each from four 20-week-old, L-dKO mice (12 tumours in total; see Methods). Liver protein components from six = 4) and two tumour samples each from four 20-week-old L-dKO mice (= 8)). For full scans, observe Supplementary Fig. 1. b, Immunoblot analysis indicates reduced LHPP manifestation (16 and 20 weeks (w)) and improved NME1 and NME2 manifestation (12, 16 and 20 weeks) in L-dKO liver (6, 12 and 16 weeks) PRKCG and tumours (20 weeks) compared to age-matched control mice (6, 12 and 16 weeks, = 4, liver cells from two mice are pooled per lane). For full scans, observe Supplementary Fig. 2. The above findings suggest that the manifestation of NME1 and NME2, but not LHPP, is definitely controlled by mTOR. To investigate this further, we acutely treated (24 h) L-dKO mice with the mTORC1 inhibitor rapamycin or the pan-mTOR inhibitor INK128. INK128 treatment reduced NME1 and NME2 manifestation in L-dKO tumours, but experienced no effect on LHPP (Extended Data Fig. 3d). Rapamycin experienced no effect on the level of NME1, NME2 or LHPP (data not shown). Thus, the manifestation of NME1 and NME2 seems to be mTORC2 dependent, whereas LHPP manifestation is definitely mTOR self-employed. The combined upregulation of NME1 and NME2 (histidine kinases) and downregulation of LHPP (putative histidine phosphatase) prompted us to investigate protein histidine phosphorylation in L-dKO tumours1,16. Phosphohistidine (pHis) is present in two isomers, 1-pHis and 3-pHis, depending on the position of the phospho-acceptor nitrogen in the histidine imidazole ring (N1 and N3 positions, respectively)2,17. Unlike the phosphoester (PCO) relationship in additional phosphoamino acids, the phosphoramidate (PCN) relationship in phosphohistidine is definitely warmth- and acid-labile, therefore making pHis hard to detect in biological samples18 This difficulty has been mainly circumvented from the recent development of monoclonal antibodies that specifically identify 1-pHis or 3-pHis2. The new antibodies were used to assess histidine phosphorylation (1-pHis and 3-pHis) in L-dKO tumours and control liver (Fig. 3a) (observe Methods). The immunoblot signals of both 1-pHis and 3-pHis were high in tumours compared AZD8055 to control cells (Fig. 3a, b). Heating sample-buffer-solubilized lysates at 95 C for 10 min before SDSCPAGE caused dephosphorylation, indicating that the signals recognized in the unheated samples are indeed histidine-phosphorylated proteins (Fig. 3a). As reported.